Whole-cell Brij protein lysis, nuclear and cytosolic protein extractions, and Western-blot analyses were performed as described [46] (link), [65] (link). Antibodies used for detection of the respective proteins and their relevant dilutions were: pSTAT5 (#9351, Cell Signaling Technology; 1∶1000), STAT5A (L-20, sc-1081, Santa-Cruz Biotechnology; 1∶1000), STAT5B (G-2, sc-1656, Santa-Cruz Biotechnology; 1∶200), STAT5A+B (C-17, sc-835, Santa-Cruz Biotechnology; 1∶1000), pJAK2 (Cell Signaling Technology, #3771; 1∶200), JAK2 (#3230, Cell Signaling Technology; 1∶500), α-tubulin (DM1A, sc-32293, Santa-Cruz Biotechnology; 1∶200), Anti-Rabbit IgG-Peroxidase (SIGMA A0545; 1∶10,000), Anti-Mouse IgG-Peroxidase (SIGMA A8924; 1∶10,000). Apparent molecular weight of detected proteins was as predicted by the antibody manufacturers, i.e. 125 kDa for JAK2, 90 kDa for STAT5 (with STAT5A running slightly slower than STAT5B in SDS-PAGE, as confirmed by the STAT5A+B immunoblots) and 55 kDa for α-tubulin. Chemoluminescence detection was performed using Amersham ECL Prime (RPN2232, GE Healthcare Life Sciences) or SuperSignal West Femto (34095, Thermo Fisher Scientific) for weaker signals, and images were captured on an ImageQuant LAS 4000 mini imaging system (GE Healthcare Life Sciences). Immunoblots shown are representative of at least three independent experiments.
Imagequant las 4000 mini imaging system
Manufactured by GE Healthcare
Sourced in United States
The ImageQuant LAS 4000 mini imaging system is a compact and versatile lab equipment designed for capturing and analyzing images of various biological samples. It utilizes a high-sensitivity CCD camera and advanced imaging technology to provide accurate and reproducible results. The system is capable of detecting and quantifying a wide range of signals, including chemiluminescence, fluorescence, and near-infrared signals, making it suitable for a variety of applications in life science research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (8 mentions)
Lysis buffer, by Beyotime (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Vimentin, by Bioworld Technology (4 mentions)
N cadherin, by Bioworld Technology (4 mentions)
β actin, by Bioworld Technology (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Multi gauge version 3, by GE Healthcare (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Zeb1 and zeb2, by Proteintech (2 mentions)
E cadherin, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
27 protocols using imagequant las 4000 mini imaging system
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Protein Samples
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Cells were washed twice with ice-cold phosphate buffer saline, harvested, and resuspended in RIPA Lysis Buffer (Beyotime, Shanghai, China) plus protease inhibitor cocktail (MedChemExpress). Cell lysates were obtained by centrifugation at 12 000 r·min−1 for 10 min at 4 °C. Equal amounts of each sample were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with the designated antibodies. Antibody-specific labeling was revealed by incubation with HRP-conjugated secondary antibodies for 1 h and visualized with an electrochemiluminescence (ECL) kit (Millipore). Images were captured and analyzed with an ImageQuant Las 4000mini imaging system (GE Healthcare Life Science, Chicago, IL, USA). All immunoblotting assays were conducted with three biological replicates, and representative images are shown.
3
Western Blot Analysis of Stress Response Proteins
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Cells were lysed in RPRA buffer (Beyotime, China), and protein concentration was measured by BCA Kit (Pierce, USA). Protein samples were separated by SDS-PAGE and subsequently transferred onto PVDF membranes. Membrane was blocked in 5% de-fat milk and incubated with primary antibody at 4°C for a night and sequentially HRP-conjugated secondary antibody at room temperature for 1 h. ImageQuant LAS 4000 mini imaging system (GE, Fairfield, USA) and Western HRP Substrate (WBLUF0500, Millipore) were used in chemiluminescent detection. The antibody used in this study are as following: anti-GAPDH (M017, TransGen), anti-PERK (5683, CST), anti-IRE1α (3294, CST), anti-ATF6 (24169-1-AP, Proteintech), anti-BiP (3177, CST), anti-Bax (2774, CST), anti-Bcl-2 (4223, CST), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz), and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz).
4
Western Blot Analysis of Cellular Proteins
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Western blot assays were performed according to the common methods. Briefly, equal amounts of protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk at room temperature for 2 h and incubated at 4 °C overnight with primary antibodies against β-actin as a control (1:500; Proteintech), α-SMA (1:200; Abcam), FSP-1 (1:1000; Abcam), FAP (1:800; Abcam), LOX (1:1000; Abcam), E-cadherin (1:1000; CST), N-cadherin (1:1000; CST), vimentin (1:500; Santa Cruz Biotechnology), β-catenin (1:1000; CST), FAK (1:1000, CST), p-FAK(Tyr397) (1:1000, CST), c-myc (1:500, CST), cyclin D1 (1:500, CST), Lamin B1 (1:1000, CST) followed by incubation with anti-goat immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan Golden Bridge Bio, Beijing, China) for 45 min at room temperature. The protein bands were detected using ECL chemiluminescence reagents (Millipore) and visualized using the ImageQuant LAS4000 Mini Imaging System (General Electrics, Louisville, Kentucky). Analyses of the bands were performed using ImageJ software.
5
Western Blot Analysis of MMP2 and MMP9
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Total protein was extracted from cells using lysis buffer (Beyotime, China). Coomassie Brilliant Blue was used to quantify the protein content. The proteins (10 μg) were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% nonfat milk in phosphate-buffered saline (PBS) containing Tween-20 (PBS-T) for 2 h at room temperature. The blots were then probed with primary antibodies specific for MMP2 (1 : 100, Boster, China), MMP9 (1 : 100, Boster, China), and β-actin (1 : 1000; Bioworld, USA) overnight at 4°C, washed twice with PBST, and incubated with horseradish peroxidase-conjugated (HRP) secondary antibodies for 1 h at room temperature. Finally, the protein bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using the ImageQuantLAS 4000 mini imaging system (General Electrics, USA). Three independent trials of each experiment were carried out.
6
Western Blot Analysis of EMT Markers
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Cells were collected and lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany) for 30 min on ice, and protein concentration was measured using a BCA Protein Assay Kit (Pierce, New York, USA). Then, samples mixed with 5x loading buffer were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA) and incubated with the corresponding primary antibody and HRP-conjugated secondary antibody. The following antibodies and dilutions were used: anti-ANXA3 (1:200, sc-101885, Santa Cruz, Dallas, USA), anti-E-cadherin (1:1000, #20874-1-AP, Proteintech, Rosemont, USA), anti-N-cadherin (1:1000, #14215, CST, Danvers, USA), anti-γ-catenin (1:1000, #2309 s, CST), anti-Vimentin (1:1000, #5741s, CST), anti-IκBα (1:1000, #4812, CST), anti-phospho-p65 (1:1000, #3033, CST), anti-tubulin (1:1000, HC101-02, TransGen Biotech, Beijing, China), HRP-conjugated goat anti-mouse IgG (1:5000, sc-2005, Santa Cruz) and HRP-conjugated goat anti-rabbit IgG (1:5000, sc-2004, Santa Cruz). Chemiluminescent detection was performed using an ImageQuant LAS 4000 mini imaging system (GE, Fairfield, USA) with Western HRP Substrate (WBLUF0500, Millipore).
7
Western Blot Analysis of EMT Markers
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Cells were lysed in RIPA buffer (Beyotime, China) and protein concentration was measured by BCA Protein Assay Kit (Pierce, USA). Protein sample mixed with loading buffer were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, USA). Membrane was blocked in 5% de-fat milk and incubated with primary antibody and HRP-conjugated secondary antibody sequentially. Following antibodies were used in this study: anti-HIPK1 (ab152109, Abcam), anti-E-cadherin (20874-1-AP, Proteintech), anti-Vimentin (5741s, CST), anti-snail (3879, CST), anti-Actin (HC201, TransGen, China), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz, USA) and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz). ImageQuant LAS 4000 mini imaging system (GE, Fairfield, USA) and Western HRP Substrate (WBLUF0500, Millipore) were used in chemiluminescent detection.
8
Purification and Western Blot of EntV Protein
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E. faecalis cultures were grown for 16 h at 37°C. The spent supernatant was then treated with 5% TCA for 48 h at 4°C. Precipitated proteins were centrifuged at 10,000 rpm for 10 min, and the supernatant was then discarded. This step was repeated twice in order to ensure that all TCA was removed prior to solubilization in 6 M urea. TCA-precipitated proteins were separated by SDS-PAGE, and Western blotting was performed using a rabbit polyclonal anti-EntV antibody at a concentration of 1:1,000 and a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at a concentration of 1:10,000. The Western blots were processed using Thermo Fisher Scientific SuperSignal West Dura extended-duration substrate and imaged using the GE ImageQuant LAS4000 mini imaging system. To generate anti-EntV serum, full-length protein was recombinantly expressed from strain CEGE1 (15 (link)). Cell lysates were run on an SDS-PAGE gel, and the overexpressed protein was excised and sent to Cocalico Biologicals, Inc., for antibody generation.
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9
Immunoblotting of Protein Samples
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Cells in 2 ml cultures with an OD600 of about 0.5 were harvested by centrifugation. Cell pellets were resuspended in 0.1 ml 1X Laemmli sample solution, sonicated, and heated at 95°C for 5 min. Then, proteins were separated by SDS/PAGE and transferred onto a PVDF membrane. To reduce non-specific binding, the membrane was blocked by incubation in 5% skimmed milk in 1X PBS containing 0.05% Tween-20 (PBS-T) for 2 h. The blocked membrane was then incubated overnight with the appropriate primary antibodies, monoclonal anti-FLAG M2 antibody (Sigma, 1:10,000) or anti-RpoA 4RA2 Mouse IgG1 (BioLegend, 1:4,000), at 4°C. The membrane was washed four times with PBS-T and then probed with appropriate secondary antibodies conjugated with horseradish peroxidase, anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, 1:20,000), and incubated for 3 h at room temperature. After washing the membrane with 1× PBS-T five times, the membrane was overlaid with ECL-SuperKine™ West Femto Maximum Sensitivity Substrate (Abbkine) for anti-FLAG immunoblotting or Miracle-Star™ Western blot detection system (iNtRON) for anti-RpoA immunoblotting, and chemiluminescence was imaged using the ImageQuant LAS 4000 mini imaging system (GE healthcare).
10
Protein Expression Analysis in Cells
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After three washes with cold PBS, total protein was extracted from cells using lysis buffer. The proteins (10 μg) were resolved using 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% nonfat milk in PBS containing Tween-20 (PBS-T) for 2 h at room temperature. The membranes were incubated at 4°C overnight with primary antibodies specific for RUNX2 (1:1000), OCN (1:1000), COL1 (1:500), VEGFR1 (1:1000), Angiogenin (1:500), ERK1/2(1:500), p-ERK1/2(1:500), p38 (1:1,000), p-p38 (1:1,000), JNK (1:500), and p-JNK (1:500). After three washes with PBST (0.5% Tween 20 in PBS), the membranes were incubated with the relevant secondary antibodies (1:1000) for 1 h at 37°C, washed and visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using the ImageQuantLAS 4000 mini imaging system (General Electrics, USA). Three independent trials of each experiment were carried out. β-actin (1:500) served as an internal control.
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